Preparation of Cardiac Extracts from Embryonal Hearts to Capture RNA–protein Interactions by CLIP

The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled. Careful consideration should be given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts. This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression. The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ. Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts. We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus. Key features • The protocol refines previous methods of cardiac extracts preparation to use for CLIP assays. • The protocol allows the quantitative RNA-seq analysis of transcripts interacting with selected proteins. • Depending on the embryonal stage, a high number of hearts can be required as starting material. • The steps are adaptable to other tissues and biochemical assays.

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.81360 The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled.Careful consideration should be given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts.This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression.The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ.Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts.We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus.

Background
The dynamic interaction between RNA and proteins controls many aspects of gene expression and disease [1,2].RNA binding proteins (RBP) have been shown to affect every aspect of RNA metabolism by positively or negatively regulating transcription and splicing, cytoplasmic export, and stability of different classes of RNAs [3].RBP can also regulate mRNA subcellular localization and promote localized translation [4].On the other hand, non-coding regulatory sequences in the mRNA, such as 5′ or 3′ untranslated regions (UTR), can reciprocally influence protein fate by controlling protein translation [5].The synergistic interplay between molecular partners often occurs through the formation of dynamic RNA and protein-containing condensates.In the nucleus, this spatial distribution offers many advantages for the processes of RNA transcription and processing since it concentrates the molecular machinery necessary for gene expression in the three-dimensional space.Notably, the importance of RNA/RBP interactions was shown to be favored by noncoding RNAs.Long non-coding RNAs (lncRNAs), in particular, can guide and influence their protein interactome in both nuclear and cytoplasmic compartments due to their structural versatility and highly context-specific expression [6][7][8][9][10][11][12].Over time, many different approaches have been developed to study RNA-protein interactions [13][14].These methodologies can be classified into two main types: protein-centric or RNA-centric.The first class is based on the possibility to precipitate a specific protein of interest pCharme RV 5′ -gcactcttccttctctccga-3′ mCharme FW 5′-ggcacagacaccaaggccag-3′ mCharme RV 5′ -gcactcttccttctctccga-3′ Gapdh FW 5′-tgacgtgccgcctggagaaa-3′ Gapdh RV 5′-agtgtagcccaagatgcccttcag-3′

Procedure
This protocol is suitable for the preparation of cellular extract from UV-crosslinked cells isolated from embryonal hearts.Cardiac tissue can be highly heterogeneous and composed of different cell types, such as cardiomyocytes, cardiac fibroblasts, and endothelial cells.Due to their size (lower than strainer cutoff), all these cells are kept during the filtering step, which is mainly required to remove clumps and tissue debris.To maintain in vivo interactions, we recommend the use of freshly collected hearts as cells need to be cross-linked while they are still viable.The cardiac extracts can be used as input for CLIP assay.An IgG negative control should always be run in parallel to the specific IP to check the specificity of the antibody (see sections D-G and [11]).It is important to note that at least 1 mg of total extract per condition (IP-specific and IgG negative control) is necessary.In Taliani et al. (2023) [11], a total of ~60 hearts (E15.5) were collected for CLIP and yielded ~5.3 mg of protein extract (0.09 mg for each E15.5 heart).

A. Embryonal hearts isolation
1. Sacrifice the pregnant mouse by CO2 or cervical dislocation as approved by the Institutional Animal Use and Care Committee.4. Carefully cut the placenta to extract the embryos.Place the embryos in a Petri dish filled with PBS to wash away the blood (Figure 1A, lower panel).5. Cut the heads of the embryos and open the chest cavity to collect the hearts (Figure 1B).Depending on the developmental stage (E10.5-E15.5),hearts can be very small (1-3 mm).6. Transfer the hearts to a 1.5 mL tube with 1 mL of cold PBS buffer.Tip: To facilitate manual dissociation (section B), do not store more than 5-6 hearts in a single 1.5 mL tube.

B. Manual dissociation
Attention: Prepare 1 mL of dissociation media.This volume is intended for 10-12 E15.5 embryonal hearts.1. Carefully remove as much PBS as possible to let the hearts settle down to the bottom of the tube (Figure 2A, left).2. Add 500 μL of cold dissociation media to each tube (5-6 hearts).
3. Mash the hearts with a pestle for 2-4 min on ice.4. Further dissociate the tissue by pipetting the solution with a 1,000 μL tip until no big clumps of tissue can be observed (Figure 2A, right).Tip: For a faster dissociation, trap the hearts between the pestle and the edge of the 1.5 mL tube (Figure 2B).It is possible to follow the dissociation state by looking through the tube against a source of light.

C. Cardiac cells preparation
1. Place the 70 μm strainer (Neonatal Heart Dissociation Kit) on a 15 mL tube on ice.2. Slowly add the cardiac homogenates to the strainer with a P1000 pipette.The solution will be filtered with gravity (Video 1).

Video 1. Filtration of cardiac homogenates using a cell strainer
3. Wash the strainer with PBS until 5 mL of volume is reached in total.
4. Use a cell lifter to facilitate the filtration process by gently scraping the top of the strainer.Be careful not to break the filter membrane (Video 2).

D. Extract preparation
1. Remove the plate lid and UV-crosslink the cells in a Spectrolinker UV Crosslinker at 254 nM with 4,000 μJ/cm 2 on ice.2. Harvest the cells using a cell strainer and transfer the cell suspension to a 15 mL tube.
3. Centrifuge at 600× g for 5 min at 4 °C.4. Remove the supernatant very gently to avoid disturbing the cell pellet and snap-freeze the cell pellet on liquid nitrogen.Pellets can be stored at -80 °C for up to 12 months.Attention: To proceed with extract preparation, scale up the number of hearts and repeat sections A-B. 5. Resuspend the frozen pellets in 3 mL of NP40 lysis buffer.6. Pipette up and down with a P1000 pipette until the solution becomes homogeneous.Split the volume evenly in three 1.5 mL tubes (1,000 μL each).7. Incubate the tubes for 10 min at 4 °C with gentle shaking.8. Perform six cycles of sonication at low intensity (see instrument manual) for 30 s at 4 °C with a Bioruptor Plus sonication device to ensure nuclear membrane lysis.9. Centrifuge at 16,000× g for 5 min at 4 °C and collect the supernatant, which represents the total cellular extract.Attention: After centrifugation, the supernatant should be clear.If the sample is still turbid, perform an additional centrifugation (16,000× g for 5 min at 4 °C) and repeat steps D5-D9 using a lower quantity of NP40 lysis buffer (1 mL instead of 3 mL).10.Quantify the protein extract concentration with Bradford assay.Then, dilute the sample in supplemented NP40 lysis buffer to adjust the final concentration to 1 mg/mL.

E. Beads loading
Attention: Prepare 1 mL of PBT supplemented with 1× PIC and 1:200/400 Ribolock.This volume is intended for the 2 h incubation of two reactions (IP and IgG).The washing volume is not included.1.For each immunoprecipitation, use two 1.5 mL tubes labeled IP (protein-specific) and IgG (negative control).Gently mix the Dynabeads Protein G magnetic particles and add 30 μL of beads to each tube.2. Wash the beads by adding 1 mL of PBT per tube.[11].Upper panel, protein analysis: western blot analysis performed on protein samples from MATR3-CLIP assay.GAPDH protein serves as a loading control.INPUT (Inp) samples represent 10% of the total protein extracts.Lower panel, RNA analysis: RT-qPCR performed on RNA samples from MATR3-CLIP assay.pCharme enrichments were compared to mCharme and Gapdh transcripts, both used as negative controls [7,11] in MATR3 Ip and IgG RNA samples.RT-qPCR quantification is expressed as percentage (%) of Input.

Data analysis
RNA samples from CLIP assays are suitable for both RT-qPCR and RNA-sequencing.For RT-qPCR, the enrichment of target RNAs can be graphed as the ratio of IP over INP.In the case of RNA sequencing, a detailed description of the analysis is presented in the Material and Methods section of Taliani et al. (2023) [11] (MATR3 CLIP-seq analysis).For the identification of reliable RBP RNA interactors, at least two biological replicates must be processed.

Validation of protocol
This protocol (or parts of it) was used and validated in the following research article(s): • Taliani et al. ( 2023) [11].The long noncoding RNA Charme supervises cardiomyocyte maturation by

General notes
1.The cell composition from heart tissue dissociation is not pure but consists of different fractions of cardiomyocytes, cardiac fibroblasts, and endothelial cells.This heterogeneity could cloud the results if your protein/RNA target is lowly expressed or expressed in an underrepresented population of cells.2. The protocol can be adapted to different starting substrates (e.g., neonatal hearts).Other tissues can also be used depending on the efficacy of viable cell purification.

5 Published: Oct 20, 2023 4 .
Proteinase K buffer (50 mL)Note: Solution can be stored at room temperature for up to six months.On the day of the experiment, add 0.5 mM DTT (1 M stock), 1× PIC, and 1:200/400 Ribolock to the needed volume.

6 Published: Oct 20, 2023 2 .
Cite as: Buonaiuto, G. et al. (2023).Preparation of Cardiac Extracts from Embryonal Hearts to Capture RNA-protein Interactions by CLIP.Bio-protocol 13(20): e4857.DOI: 10.21769/BioProtoc.4857Wet the skin and the fur of the mouse with 70% ethanol to avoid samples contamination with mice hair.3.Position the mouse under the hood.Pinch the skin with tweezers, pull up and incise and pull apart the skin to expose the abdomen.Cut the peritoneum: you will see all the embryos contained in the placenta (Figure1A, upper panel).

Figure 1 .
Figure 1.Collection of developing hearts from mouse embryos.A. Representative image of mouse (E15.5)embryos enveloped in the placenta sack.Isolated single embryos are shown below.B. Zoom-in image of representative E15.5 mouse embryo and heart.Ruler is shown for measurement assessment.

Figure 2 .
Figure 2. Preparation of cardiac extract.A. 1.5 mL tubes containing E15.5 hearts (n = 5) resuspended in PBS before (left) and after (right) manual dissociation with a pestle.B. Graphic representation of the procedure used to speed up dissociation by trapping the embryonal hearts between pestle and tube.

Figure 1 AB 7 Published
Figure 1 A B

8 Published: Oct 20, 2023 5 .
Cite as: Buonaiuto, G. et al. (2023).Preparation of Cardiac Extracts from Embryonal Hearts to Capture RNA-protein Interactions by CLIP.Bio-protocol 13(20): e4857.DOI: 10.21769/BioProtoc.4857Pour the 5 mL of filtered cardiac homogenates in one Petri dish (100 mm of diameter) at room temperature and check cells' viability.Tip: Slightly move the plate, and consequently the media, to better visualize the translucent cells under a brightfield light microscope.Cells should stay in suspension (Figure3).To visualize the cardiac cells, fluorescent or colorimetric dyes can also be used on a small volume of the filtered homogenates.

Figure 3 .
Figure 3. Cardiac cells preparation.Representative image of freshly isolated cells before UV-crosslinking step.Black arrows indicate examples of viable cells.

Figure 4 .
Figure 4. Analysis of CLIP results [adapted from Taliani et al. (2023)[11].Upper panel, protein analysis: western blot analysis performed on protein samples from MATR3-CLIP assay.GAPDH protein serves as a loading control.INPUT (Inp) samples represent 10% of the total protein extracts.Lower panel, RNA analysis: RT-qPCR performed on RNA samples from MATR3-CLIP assay.pCharme enrichments were compared to mCharme and Gapdh transcripts, both used as negative controls[7, 11]  in MATR3 Ip and IgG RNA samples.RT-qPCR quantification is expressed as percentage (%) of Input.

Problem 1 :
The quantity of cells obtained from the hearts is low Possible cause: Low starting material Solution: Prepare and freeze multiple cell pellets Problem 2: The solution does not flow through the 70 μm strainer Possible cause: The remaining cell clumps in the sample clog the strainer Solution: Recover the solution from the strainer, use a micropipette to further disrupt cell clumps, and add PBS to dilute the sample.Load the solution in the strainer, keeping the micropipette perpendicular to the strainer membrane, and apply some pressure on the filter without breaking the membrane.